PAT December, 2014; 10 (2): 54-64; ISSN: Online copy available at

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1 PAT December, 2014; 10 (2): 54-64; ISSN: Online copy available at Toxicity and Repellency Action of Lantana camara on Annopheles gambiae Kemabonta 1 *, K, A, Aninye 1, A., Ajelara 2, K.O. and Adu. B. W 3 1 Department of Zoology, Faculty of Science, University of Lagos, Lagos State Nigeria 2 Department of Zoology, and Environmental Biology, Faculty of Science, Lagos State University, Lagos. Nigeria 3 Department of Biological Sciences, Ondo State University of Science and Technology, Okitipupa, Ondo State * correspondence author: kennykemabonta@yahoo.com Abstract Larvicidal, Adulticidal and repellency of Lantana camara was investigated against Anopheles gambiae. 2 nd instar larvae and adult of Anopheles gambiae were exposed to serial dilution of the stock solution in 10, 20, 30, 40 and 50%v/v of hexane and ethanol extracts of L camara. The control experiment contained distilled water only. Mortality rate of the larvae and adults were calculated after every 24hrs for three days. Repellency and knockdown action was determined using improvised mosquito coil. Hexane and Ethanol extracts were effective against An gambiae. Hexane extract had a higher larvicidal effect while ethanol had a higher adulticidal and repellency effect than hexane. An gambiae was more susceptible to hexane than ethanol extract of L. camara. It was 2.41(24hours), 3.20 (48 hours) and 1.96 (at 72hours) times more susceptible than ethanol. Protection (repellency) increased with increase in concentration of both hexane and ethanol extracts of L. camara. 100% protection was achieved with 100% v/v concentration of both hexane and ethanol respectively. For the coil, percentage knockdown increased with increase in concentration of L. camara powder. Highest knockdown 90% was also achieved with 100% concentration of L. camara coil Keywords: Adulticidal, larvicil, repellency, mosquito coil, hexane extract, ethanol extract, Lantana camara, Anopheles gambiae Introduction Malaria is a mosquito borne infectious disease of humans and other animals caused by a single celled protozoan parasiteplasmodium. Anopheles gambiae transmits Plasmodium falciparum, which is the most severe of the four malarial agents. There are an estimated 300 to 500 million cases of malaria each year and as a result, 1.5 to 2.7 million deaths occurs worldwide. Malaria is a major cause of infant mortalityin tropics and sub-tropics regions of the world. It is the only insect borne parasitic disease comparable in impact to the world's major killer transmissible diseases: diarrhea, acute respiratory infections, tuberculosis and AIDS (Denloye et al., 2003). Synthetic insecticides have been used to control mosquitoes over time, but this leads to adverse repercussions such as poisoning, environmental destruction and development of resistance in target species (Don-Pedro, 1980; Boroffice and Borofice 1993). These problems have necessitated the need for search and development of environmentally

2 PAT 2014; 10 (2): 54-64: ISSN: ; Kemabonta et al;: Toxicity and Repellency Action of Lantana 55 safe, biodegradable, low cost, indigenous methods for malaria vector control which can be used as an alternative to synthetic insecticides (Balandrin et al., 1985). The most important of alternatives to synthetic insecticides is Phytochemicals (plant extracts) with a broad range of activities against mosquitoes such as ovicidal, larvicidal, adulticidal, oviposition deterrence, developmental toxicity, antifeedants, repellents, hatching and emergence blockers (Bakkali et al., 2008, Sukumar, 1991).Though lots of research works have been carried out against natural products on mosquitoes (Bowers et al., 1995, Denloye et al., 1995, 2003, Dua, 2010). There are still lots of medicinal plant products that are yet to be tested fortheir potency against the control of mosquitoes. Lantana camara, also known as big sage (Malaysia), wild sage, red sage, white sage (Caribbean) and tick berry (South Africa) and Elepo (in Yoruba:Nigerian language) is a species of flowering plant within the family Verbenaceae. The stalks of L. camara have been used in the construction of furniture such as chairs and tables, however, the main uses have historically been medicinal and ornamental. Studies show that L. camara has antimicrobial, fungicidal and insecticidal activity against stored grain insect pests, vegetable crops pest, mosquito larvae, repellent, and other biological activities.l. camara has also been used in traditional herbal medicines for treatment of ailments, including cancer, skin itches, leprosy, rabies, chickenpox, measles, asthma and ulcers (Balandrin et al., 1995). The aim of the study is to evaluate the repellency and larvicidal effect of hexane and ethanol extracts of Lantana camara against adult and larvae Anopheles gambiae mosquito respectively and the repellency effect and knockdown action of dried powdered extract of the leaves as impoverished coil on adults of Anopheles gambiae. Finally, to determine which extract (hexane and ethanol) has a higher toxicity effect on Anopheles gambiae. Materials and Methods Source and Preparation of Test Plant Species Fresh leaves of Lantana camara were obtained from ornamental gardens in University of Lagos (UNILAG) (Plates1and 2). The specimen of the plant was identified as L. camara UNILAG herbarium. The leaves were washed under running water and air dried at room temperature on laboratory tables for 7days. The dried leaves were pulverized using an electronic blenderand thereafter, stored in air tight polythene (Ziploc bags ) bags until needed.

3 PAT 2014; 10 (2): 54-64: ISSN: ; Kemabonta et al;: Toxicity and Repellency Action of Lantana 56 Plate 1Lantana camara plant with its yellow flower from University of Lagos Plate2 Cluster of black berry fruits of Lantana camara from University of Lagos Extraction of Hexane and Ethanol Extracts of L. camara Leaf Powder Ethanolic and hexane extracts of L. camara were carried out on the leaf powder. 10 g of dried Lantana camara leaf powder was steeped in 150 ml of ethanol in a 250 ml conical flask properly cocked to avoid evaporation for 24 hours at room temperature. The mixture was stirred thoroughly and filtered through Whatman filter paper size 10. The filtrate was kept in Amber bottles until needed. The procedure was repeated several times to obtain 250 ml for each crude extract. It was labeled and stored for use in bioassay studies. Same procedure was used for hexane extraction of Lantana camara. Preparation of Mosquito Repellent Coil from L. camara Leaf Powder Insecticidal repellent coil were prepared from the leaf powder of L. camara that has been dried at room temperature. The leaves were pulverized with pestle and mortar and further pulverization into a powder form was achieved with a Kenwood electric kitchen blender. Different concentrations of the coil were achieved by moulding with saw dust as inert material blended with starch made from processed cassava as binder. Concentrations of 20, 40, 60, 80 and 100% were made in ratio formula by adding 2 g of the test plant with 18 g of saw dust to obtain 20%; and graduated ratio for 40, 60, 80 and 100%. Moulded coils were dried in the laboratory table laid with foil paper (Plate 3) at room temperature. Moulded saw dust shavings bounded with processed starch from cassava was used for the control treatment.

4 PAT 2014; 10 (2): 54-64: ISSN: ; Kemabonta et al;: Toxicity and Repellency Action of Lantana 57 Plate3: A picture showing molded coil of Lantana camara leaf powder Larva and Adult Rearing Anopheles gambiae larvae were collected from the National Institute of Medical Research (NIMR) Yaba, Lagos. The larvae (over 200) were placed in plastic containers (5 plastic containers of 5 litres each) that contained yeast powder in water until they emerged into adults. The adults were used for the adulticidal experiments. The emerged adults were periodically blood fed on volunteer s arms dipped into the cage through the netting and were also were provided with 10% glucose solution on cotton wicks to feed on. The cotton was kept moist and changed every day. Toxicity of the Ethanol and Hexane Extract of L. camara on An. gambiae Larvae Ten (10) 2 nd instar Anopheles gambiae larvae were transferred into a transparent disposable cup. The larvae were then exposed to serial dilution of the stock solution in 10, 20, 30, 40, 50% v/v. The control experiment contained distilled water only. The mortality rate of larvae was recorded after every 24hrs for three days at ambient temperature and relative humidity. This procedure was replicated three times. Percentage mortality as calculated thus; Total number of dead larvae x 100 Total number of larvae Repellency and Knockdown Effect of Ethanol and Hexane Extract of Lantana camara Leaf Powder against Adult Anopheles gambiae The adult Anopheles gambiae used in the test were 2 days old. Two cages were used and ten mosquitoes were put in each cage. The crude extract was serially diluted with distilled water at 20, 40, 60, 80 and 100% concentrations. Control was also set using only distilled water. The arms of the volunteer were initially washed with toilet soap

5 PAT 2014; 10 (2): 54-64: ISSN: ; Kemabonta et al;: Toxicity and Repellency Action of Lantana 58 and distilled water. Ethanol or hexane in cotton wool was used to clean the volunteer s arms to prevent the mosquito s attractiveness to the sweat of the arm. One of the cleaned arms of the volunteer was rubbed with each level of concentrations of Lantana camara leaf powder at each time of treatment application while the second hand was rubbed with distilled water (control). The two arms were each exposed to ten 0-2 day old unfed adult mosquitoes at thirty minutes interval. The repellency of the different concentrations of the extract was measured on the basis of the number of bites received within thirty minutes on both arms. After each exposure for the different concentration levels, the arm was thoroughly washed and cleaned with ethanol (for ethanol extract) and hexane (for hexane extract) before another dosage was introduced on the arm into another cage. New unfed adult mosquitoes were used for each concentration levels. The Anopheles gambiae adult used in the test was 0-2 day old. Two cages were used and ten mosquitoes were put in the cage each. For the coil, varying concentrations of 20, 40, 60, 80 and 100% and control were molded; mosquitoes were exposed to smoldering coil and repellency at different concentration was recorded every 30 minutes. The repellency of the different concentration of the coil was measured on the basis of the number of bites on the arms within thirty minutes while the knockdown effect was calculated on the number of insects that lay (fallen down) on the floor of the cage. The percentage repellency and knockdown effect was calculated as thus: Mean no of bites received in control area (arm) Mean number of bites received - mean number of bites received in treatment area (arm) x 100 or and for the knockdown effect: Mean no of fallen insects in the control area - mean number of fallen insects in the treatment area x 100 Mean number of fallen insects Statistical Analysis LC50 for 24, 48 and 72 hours (for larvae) was calculated using the Probit analysis after Finney (1971). The control mortalities were corrected by using Abbott s formula (Abbott s 1925). Corrected mortality = Observed mortality in treatment observed mortality in control x Control mortality

6 PAT 2014; 10 (2): 54-64: ISSN: ; Kemabonta et al;: Toxicity and Repellency Action of Lantana 59 The LC5, LC50, LC99 and 95% upper confidence limit (UCL) and lower confidence limit, regression and chi-square values were calculated by using Probit analysis (Finney 1971). Data were subjected to statistical analysis using SPSS 20. The different levels of concentration of hexane, ethanol, repellency and knock down at various times were analyzed separately using analysis of variance (ANOVA). Means were compared using Duncan s Multiple Range Test at 0.05% level of significance. Results Effect of hexane and Ethanol Extract of L. camara Leaf Powder on An. gambiae Larval mortality of An. gambiae increased with increase in concentration in both extracts as well as the exposure time (Tables 1 and 2).Larval mortality with hexane extract ranged from 73.3 at 24hours to 100.0% at 72hours (Table 1) and from 53.3 to 80.0% respectively with ethanol extract (Table 2). All the larvae (100%) at 30 to 50% v/v hexane extract died after 72hours and, were significantly higher than mortality in control and 10%v/v hexane extract (Table 1). Mortality in ethanol extract was 80 to 93% for adult but larvae died at same concentration and were also significantly higher than control and 10% v/v extract. Table 1: The toxicity effect of hexane extract of Lantana camara leaf powder on larvae of Anopheles gambiae Time of Exposure Concentration (%v/v) 24 hours 48 hours 72 hours Control 0.00±0.00 a 0.00±0.00 a 0.00±0.00 a ±3.06 ab 53.33±1.15 b 80.00±2.00 b ±3.46 ab 60.00±2.00 bc 86.67±1.15 bc ±2.31 b 86.67±1.15 cd ±0.00 c ±2.00 b 93.33±1.15 d ±0.00 c ±2.31 b 86.67±2.31 cd ±0.00 c Mean values with different subscript on the same column show significant differences at p<0.05. (Df = 5)

7 PAT 2014; 10 (2): 54-64: ISSN: ; Kemabonta et al;: Toxicity and Repellency Action of Lantana 60 Table 2 The toxicity effect of ethanol extract of Lantana camara leaf powder on larvae of Anopheles gambiae Time Concentration (%v/v) 24 hours 48 hours 72 hours Control 0.00±0.00 a 0.00±0.00 a 0.00±0.00 a ±0.00 a 6.67±1.15 a 46.67±2.31 b ±1.15 a 26.67±2.31 a 60.00±2.00 bc ±3.46 ab 60.00±3.46 b 86.67±1.15 cd ±1.00 bc 60.00±1.15 b 93.33±1.15 d ±1.15 c 60.00±1.51 b 80.00±2.00 cd Mean values with different subscript on the same column show significant differences at p<0.05. (Df = 5) Mortality Rate of Hexane and Ethanol Extract of Lantana camara Leaf Powder on Anopheles gambiae Larvae Mortality rate of hexane and ethanol extracts of L. camara is shown in figures 1 to 6. In both extracts, mortality rises, gets to the peak then drops (Figures 1,3,4,6) or remains constant (Figure 5). Figure 1: Mortality rate of hexane extract of Lantana camara within 24hrs. Figure 2:Mortality rate of ethanol extract of Lantana camara within 24hrs

8 PAT 2014; 10 (2): 54-64: ISSN: ; Kemabonta et al;: Toxicity and Repellency Action of Lantana 61 Figure 3: Mortality rate of hexane extract of Lantana camara within 48hrs Figure 4:Mortality rate of ethanol extract of Lantana camara within 48hrs Figure 5:Mortality rate of hexane extract of Lantana camara within 72hrs Figure 6: Mortality rate of ethanol extract of Lantana camara within 72hrs LC50 Values of Hexane and Ethanol Extracts of Lantana camara Leaf Powder on Larvae of Anopheles gambiae The LC50 value of hexane on Anopheles gambiae larvae was significantly lower than that of ethanol extract. It ranged from (72hours) to (24hours) for hexane extract and from (72hours) to (24hours) for ethanol extract (Table 3-5). The toxicity factor also shows that Anopheles gambiae is more susceptible to hexane extracts than ethanol extract of L. camara leaf powder at the following differing ratios: 2.4 (24 hours) > 3.20 (48 hours) > 1.96 (72 hours).

9 PAT 2014; 10 (2): 54-64: ISSN: ; Kemabonta et al;: Toxicity and Repellency Action of Lantana 62 Table 3: LC50 values of hexane and ethanol extract of Lantana camara Leaf powder on Anopheles gambiae at 24 hrs. Test botanical Duration LC50 Confidence interval Regression equation Slope S.E DF TF Hexane 24hrs Y= (-3.823)x Ethanol 24hrs Y= (-4.946)x Table 4: LC50 values of hexane and ethanol extract of Lantana camara Leaf powder on the larvae Anopheles gambiae at 48 hrs. Test botanical Duration LC50 Confidence interval Regression equation Slope S.E DF TF Hexane 48hrs Y= (-2.982)x Ethanol 48hrs Y= (-5.197)x Table 5: LC50 values of hexane and ethanol extract of Lantana camara Leaf powder on Anopheles gambiae larvae at 72 hrs. Test botanical Duration LC50 Confidence interval Regression equation Slope S.E DF TF Hexane 72hrs Y= (-2.070)x Ethanol 72hrs Y= (-3.124)x Repellency Test of Hexane and Ethanolic Extracts of Lantana camara Leaf Powder and Knock Down Effect of Molded Coil on Anopheles gambiae Adults After 30 Minutes Repellency of L. camara on adult An. gambiae mosquitoes is shown in Table 6. The knockdown effect increased with concentration in both hexane and ethanol extracts of L. camara leaf powder. 100% protection was achieved with 100% v/v concentration of both hexane and ethanol extracts, respectively. Highest knockdown effect of 90% of test insect was recorded at 100% v/v concentration (Table 6).

10 PAT 2014; 10 (2): 54-64: ISSN: ; Kemabonta et al;: Toxicity and Repellency Action of Lantana 63 Table 6: The repellency and percentage knockdown effect of of L camara on Adults of An. gambiae adult Repellency (%) Percentage Knock Down Conc(%v/v) Hexane Ethanol Coil Control Discussion and Conclusion The study showed that the crude extracts of L. camara leaf powder were effective in controlling the larvae of An. gambiae. This is in agreement with the findings of Nath et al., (2006) who found that the leaf extract of L. camara showed larvicidal effectiveness against C. quinquefasciatus and Aedes albopictus. Moreover, Innocent et al., (2008) in their study found the root barks extracts of Lantana viburnoides to be toxic against 3 rd and early 4 th instar larvae of Anopheles gambiae. They reported that extracts could serve as a source of larvicides for managing various mosquito habitats in the field even in their semi purified form. The presence of compounds such as lantadene, triterpenoids and furanonaphthaquinones in Lantana species maybe the cause of the plant s larvicidal properties on mosquitoes (Nath et al., 2006 and Innocent et al., 2008). The extracts from L. camara leaves also showed adulticidal activity against An. gambiae using hexane (80%) and ethanol (100%) extracts respectively. This is in agreement with Dua et al (2010) who found the adulticidal activity of the oil of L camara to be most toxic to An. fluviatilis followed by An. culicifacies, C. quinquefasciatus, An. stephensi and Ae. Aegypti. Moreover, hexane extract showed a higher adult mosquito repellency and larvicidal effect (100 and 90% respectively) than ethanol extract on Anopheles gambiae. This study also shows that the crude extracts of hexane and ethanol of Lantana camara leaf powder are slow-acting botanicals. This is because it was at 72 hours post treatment that the effect of mortality rose up to 100% in hexane and 93.3% in ethanol. While at 24 hours the efficacy of the extracts showed significantly lower mortality of 73.3 and 53.3%, respectively for hexane and ethanol extracts. LC 50 s of the leaf extracts showed Lantana camara to be promising as larvicides, adulticides and repellents against Anopheles gambiae.

11 PAT 2014; 10 (2): 54-64: ISSN: ; Kemabonta et al;: Toxicity and Repellency Action of Lantana 64 The use of the plants in insect control offers a safer alternative to synthetic chemicals and can be obtained by individuals and communities easily at a very low or no cost at all. Lantana camara leaves are weed plants that grow around in many places in Nigeria. The leaves can be used as a coil to repel mosquitoes in such areas. Since lantana camara is active against insects, less expensive, easily biodegradable to non-toxic products it can be incorporated into integrated vector control. References Bakkali, F., Averbeck, S., Averbeck, D. and Idaomar, M. (2008).Biological effects of essential oils; a review of recent research.foodand Chemical Toxicology 46 (2): Balandrin, M. F., Klocke, J. A., Wurtele, E. S. and Bollinger, H. (1985).Natural plant chemicals; sources of industrial medicinal materials.science 228: Boroffice, O. B. and Boroffice R. A. (1993).Pesticide usage in Nigeria and the health implication.nigeria Journal of Research and Review in Science 1: Denloye A.A, Ajelara K.O, Oluwa O.K, Eshinlokun A.O and Makanjuola W.A. (2009).Insecticidal activity of petroleum ether extract and essential oil of Chenopodiumambrosioidies L. (chenopodium) against Anopheles gambiae.actaentomologicasina 52(8): Denloye, A. A., Makanjuola W. A. and Babalola, O. O. (2003).Toxicity and repellency of crude aqueous extracts of garlic (Allium sativum) on larval and adult Anopheles mosquitoes.african Entomology 11 (2): Don - Pedro K. N. (1980). A population explosion of Aphis crassivora Koch following DDT aplicaation in a cowpea plot (Vignaunguiculata) in Nigeria. Journal of Natural History 14: Dua V. K., Pandey A. C., and Dash A. P. (2010). Adulticidal activity on essential oil of Lantana camara leaves against mosquitoes. Indian J. Medical Research 131: Innocent E., C. C. Joseph, N. K., Gikonyo, M. J., Moshi M., H.H. Nkunya and A. Hassanali (2008). Mosquito larvicidal constituents from Lantana viburnoidesspviburnoidesvarkisi(a. rich) Verdc (Verbenaceae). J Vector Borne Dis. 45, Nath D. R., M. Bhuyan and S. Goswami (2006).Botanicals as Mosquito Larvicides.Defence Science Journal. 56: Sukumar, K. M., Perich, and Boobar, L. (1991). Botanical derivatives in mosquito control: a review. Journal of America Mosquito Control Association 7: